The synaptic cytoskeleton plays a critical role in the formation and maintenance of synapses in the central nervous system. Recent studies have identified a new protein motif called a PDZ domain which may be important in the proper targeting of proteins to cell-cell junctions. PDZ domains within cytoskeleton associated proteins mediate the interaction of the cytoskeleton with the C-termini of a variety of membrane proteins. The PDZ domains of the PSD/SAP family of proteins have been implicated in the synaptic targeting of NMDA receptors and K+-channels in neurons. We have recently identified novel PDZ domain-containing proteins, GRIP1 and 2 (Glutamate Receptor Interacting Protein), that specifically interact with the C-termini of AMPA receptors, the major excitatory neurotransmitter receptors in the brain. GRIP1 and 2 contain seven distinct PDZ domains and appear to serve as adapter proteins to link AMPA receptors to other neuronal proteins and may be critical for the regulation of AMPA receptor function. In this research proposal we plan to further characterize the structure and function of GRIP1 and 2 and related proteins, and determine their role in the synaptic targeting of AMPA receptors. Specifically, we will identify proteins that interact with GRIP1 and 2 (GRASPs-GRIP associated proteins) to form a PDZ domain- based complex at synapses. In addition, we will examine the functional effect-of GRIP1 and 2 on AMPA receptor ion channel function using patch clamp techniques. Dominant negative constructs of GRIP1 and 2, and GluR2 will be used to disrupt the interaction of AMPA receptors with GRIP1 and 2 in neurons to investigate the functional role of GRIPs in AMPA receptor targeting. Finally, gene targeting techniques will be used to knockout the GRIP1 and 2 genes to determine their role in the development, maintenance, and regulation of excitatory synapses. This research will elucidate basic molecular mechanisms underlying synaptic transmission in the brain. Such knowledge is essential for understanding the function of the brain in health and disease.